Oligonucleotide probes to L-AP4 sensitive glutamate receptor sequences

ABSTRACT

Metabotropic receptor mGluR7 is identified and sequenced. The mGluR7 receptor subfamily mediates inhibition of transmitter release at selected glutamatergic synapses. The receptors, mGluR7-specific peptides and antibodies thereto are used to identify agonists and antagonists of G protein coupled glutamate receptor mediated neuronal transmitter release, as well as in methods of diagnosis and therapy.

GOVERNMENT SUPPORT

This invention was made in part by research grants received from theNational Institutes of Health. The U.S. government may have certainrights in the invention.

This is a division of application Ser. No. 08/176,401 filed Dec. 30,1993, now abandoned.

BACKGROUND OF THE INVENTION

Glutamate is a major excitatory neurotransmitter in the mammaliancentral nervous system. The neurotransmitter activity of glutamate isprimarily mediated by ligand-gated ion channels. The observation thatglutamate also induces responses mediated by second messengers has ledto the discovery of a distinct group of glutamate receptors coupled to Gproteins, termed metabotropic receptors (mGluRs). Schoepp and Conn,Trends Pharmacol. Sci. 14: 13-20 (1993). The first described action ofthe glutamate metabotropic receptors was inositol phospholipid (PI)hydrolysis. Nicoletti et al., J. Neurochem. 46: 40-46 (1986) andSugiyama et al., Nature 325: 531-533 (1987). Molecular cloningtechniques have revealed a large family of metabotropic receptors withdistinct transduction mechanisms, patterns of expression andsensitivities to glutamate agonists. Schoepp and Conn, supra.

Consistent with the molecular heterogeneity observed for themetabotropic receptors, electrophysiological studies have suggesteddiverse roles for these receptors in synaptic plasticity, presynapticinhibition and regulation of cell excitability by ion channelmodulation. Bashir et al., Nature 363: 347-363 (1993); Linden et al.,Neuron 7: 81-89 (1991); Baskys and Malenka, J. Physiol. (Lond.) 444:687-701 (1991); Charpak et al. Nature 347: 765-767 (1990); and Lesterand Jahr, Neuron 5: 741-749 (1990). However, the specific mGluRreceptors mediating these cellular functions are largely undefined.

Evidence for a physiological role for specific mGluR subtypes has beenderived from work with selective agonists and antagonists of thereceptors. For example, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylicacid (ACPD) is a selective and potent activator of the mGluR1, mGluR2,mGluR3 and mGluR5 receptors. Masu et al., Nature 349: 760-765 (1991);Abe et al., J. Biol. Chem. 267: 13361-13368 (1992); Tanabe et al.,Neuron 8: 169-179 (1992); and Tanabe et al., J. Neurosci. 13: 1372-1378(1993). L-2-amino-4-phosphonobutryic acid (L-AP4) has been shown toactivate mGluR4 and mGluR6. Id., Thomsen et al., Eur. J. Pharmacol. 227:361-362 (1992); Nakajima et al., J. Biol. Chem. 268: 11868-11873 (1993).L-AP4 inhibits transmitter release and voltage-dependent calcium entryin selected brain and spinal cord neurons. Koerner and Cotman, BrainRes. 216: 192-198 (1981); Trombley and Westbrook, J. Neurosci. 12:2-43-2050 (1992); and Sahara and Westbrook, J. Neurosci. 13: 3041-3050(1993). But in retinal bipolar neurons, postsynaptic L-AP4 receptorsactivate a phosphodiesterase. Nawy and Jahr, Nature 346: 269-271 (1990).

Multiple mGluR subtypes can be present within the same group of neurons.As the cellular and subcellular localization of specific mGluRs may beimportant in shaping incoming sensory information, it is important toidentify other receptors of the mGluR group. Once identified, specificagonists and antagonists can be prepared to modulate the responsesassociated with the receptor. Quite surprisingly, the present inventionidentifies a L-AP4 sensitive receptor that modulates transmitter releasein neurons that express neither mGluR4 nor mGluR6, and fulfills otherrelated needs.

SUMMARY OF THE INVENTION

The present invention provides novel isolated and purified metabotropicmGluR proteins referred to as mGluR7. The proteins may bind glutamateand induce cytoplasmic signal transduction. Allelic variants andmutations of mGluR7 proteins are included.

Also provided are isolated polynucleotides encoding mGluR7 and probes tothe polynucleotides. The polynucleotides may be present in expressioncassettes of the present invention that are useful for cellulartransformation. Such transformed cell lines are also provided by thepresent invention. Transformed cells may be employed in methods foridentifying compounds that alter mGluR7 metabolism.

Antibodies to mGluR7 receptor proteins are also provided by the presentinvention. The antibodies may be polyclonal or monoclonal. Theantibodies may be employed to detect the presence of mGluR7 inbiological samples, such as tissue homogenates, biological fluids, orcell surfaces.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-1, 1-2 and 1-3 collectively depict the nucleotide sequence (SEQID NO:1) of a mGluR7 cDNA and the deduced amino acid sequence encodedthereby. The complete nucleotide sequence of a representative mGluR7cDNA contains an open reading frame of 2745 bp and a translationinitiation consensus sequence that surrounds the presumed initiatormethionine. The solid line indicates the predicted signal peptide. Thededuced amino acid sequence is 915 amino acids. The seven transmembranesegments (I-VII) were assigned based on hydrophobicity analysis.Asterisks mark putative N-glycosylation sites while a putative CaMkinase II phosphorylation site is indicated by a filled circle.

FIG. 2 depicts the inhibition of forskolin-stimulated cAMP production bymGluR7 in baby hamster kidney cells. BHK cells transfected with mGluR7were preincubated for 20 minutes in IBMX (1 mM), then incubated for 10minutes with forskolin (10 μM) in the presence or absence of agonists orantagonists. L-AP4 (1 mM) produced the largest inhibition whileglutamate (1 mM), quisqualate (0.5 mM) and ACPD (1 mM) were lessefficient. L-AP3 (1 mM) or MCPG (1 mM) did not antagonize the inhibitionproduced by L-AP4.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides an isolated and purified proteinreceptor, designated "mGluR7," which is a member of the family of Gprotein coupled membrane receptors for the neurotransmitter glutamate.mGluR7 participates in the regulation of neurons, includingneuroendocrine neurons, and particularly mitral/tufted neurons of theolfactory bulb. mGluR7 acts possibly by mediating inhibition oftransmitter release at selected glutamatergic synapses. mGluR7 has anexpression pattern in the central nervous system (CNS) distinct fromother metabotropic receptors. The mGluR7 receptor can be coexpressedwith other receptors of the mGluR family in single neurons, and thus thecellular effects of mGluR activation may result from the integratedaction of several receptor subtypes that include mGluR7. Consequently,agonists and antagonists of the mGluR7-ligand interaction andmGluR7-mediated metabolism are important in regulating mGluR7-mediatedneurotransmitter release. Isolated polynucleotides, including the cDNAwhich encodes mGluR7, are also described as part of the presentinvention, thereby providing means to conveniently produce isolatedmGluR7 protein and to identify agonists and antagonists of themGluR7-ligand interaction and mGluR7-mediated metabolism.

The isolated mGluR7 is meant to refer to mGluR7 which is found in acondition other than its native environment, such as apart from a neuronof the CNS. This includes, for example, substantially pure mGluR7 asdefined below. More generally, isolated is meant to include mGluR7 as aheterologous component of a cell or other system. For example, mGluR7may be expressed by a cell transfected with a DNA construct whichencodes mGluR7 and which expresses other selected receptors or secondmessenger components. In another example described below, mGluR7 isexpressed by a non-neuronal cell which has been co-transfected with agene encoding mGluR4 receptor. Thus, in this context, the environment ofisolated mGluR7 is not as it occurs in its native state, particularlywhen it is present in a system as an exogenous component.

mGluR7 is meant to include any protein either derived from a naturallyoccurring mGluR7, or which shares significant structural and functionalcharacteristics peculiar to a naturally occurring mGluR7. Such areceptor may result when regions of a naturally occurring receptor aredeleted or replaced in such a manner as to yield a protein having asimilar function. Generally, it is desirable to conserve amino acidsequences associated with ligand binding. Conserved amino acidsassociated with the ligand-binding region of mGluR7 may include aminoacids 72, 157-163, 180-184, 197, 410, and 472 of FIG. 1 (SEQ ID NO:1).mGluR7 transmembrane domains, which may include amino acid segmentsI-VII of FIG. 1, typically are more tolerant of amino acid mutationsthan N-terminal (extracellular) or C-terminal (cytoplasmic) domains.Preferred substitutions, especially in transmembrane domains, include,e.g., conservative amino acid substitutions and substitutions of aminoacids from corresponding positions in other receptor molecules havingseven transmembrane segments, particularly other mGluRs.

Homologous sequences, allelic variations, and natural mutants; inducedpoint, deletion, and insertion mutants; alternatively expressedvariants; proteins encoded by DNA which hybridize under high stringencyconditions to nucleic acids which encode naturally occurring mGluR7;proteins retrieved from naturally occurring materials; and closelyrelated proteins retrieved by antisera specific for mGluR7 proteins orpeptides are also included. By "homologous" is meant sequences that haveat least about 85% homology, preferably at least 90% homology, and morepreferably at least about 95% or more homology to the amino acidsequence of a naturally occurring mGluR7 and retains the ability to bindglutamate or transduce intracellular signals.

With the mGluR7 and cDNA clones thereof provided herein, nucleotide andamino acid sequences may be determined by conventional means. Seegenerally, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2ded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, incorporated by reference herein. The invention provides isolatedand cloned mGluR7 coding sequences which are capable of expressing themGluR7 protein. By "isolated," it is meant that the molecules areremoved from their natural genetic milieu. Thus, the invention providesmGluR7-encoding DNA molecules free of other genes with which they areordinarily associated. In particular, the molecules are free ofextraneous or unwanted coding sequences, and in a form suitable for usewithin genetically engineered protein production systems. Arepresentative mGluR7-encoding cDNA is shown in SEQ ID NO:1.

Those skilled in the art will recognize that equivalent sequences couldbe prepared by substituting alternative codons. The present inventionincludes these equivalent sequences, as well as additional sequencesthat specifically hybridize to naturally occurring or equivalentsequences. Such additional sequences will hybridize to SEQ ID NO:1, orequivalents thereof, under conditions of high moderate stringency, i.e.,conditions that differentiate related molecules from background. Thoseskilled in the art will recognize that lower stringent conditions serveto identify sequences encoding functionally equivalent polypeptideshaving common structural features (e.g., allelic variations). Forexample, conditions of moderate stringency for probes of 100 or more areprewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) andhybridization conditions of 50° C. in 5 X SSC overnight (Sambrook etal., supra, previously incorporated herein by reference). Conditions ofhigher stringency may be utilized by increasing temperature ordecreasing the salt concentration of the hybridization solution.Determination of stringency hybridization conditions is within the levelof ordinary skill in the art.

Genomic or cDNA sequences encoding mGluR7 and homologous receptors ofthis subfamily of GluGR receptors can be obtained from librariesprepared from other species according to well known procedures.Complementary DNA encoding mGluR7 may be obtained by constructing a cDNAlibrary from mRNA from, for example, olfactory bulb tissue. The librarymay be screened by transcribing the library and screening the cloneswith a complementary labeled oligonucleotide probe prepared inaccordance with the mGluR7 nucleotide sequences set forth herein. Aswill be recognized by those skilled in the art, the rat mGluR7 sequencedisclosed herein is a useful tool for cloning correspondingpolynucleotides from other species. For instance, using oligonucleotideprobes prepared from rodent mGluR7, such as whole length cDNA or shorterprobes of at least about fourteen nucleotides to twenty-five or morenucleotides in length, and often as many as 40 to 50 nucleotides,prepared from areas of cDNA sequences specific for mGluR7 when comparedto other known members of the mGluR family, the mGluR7 of other species,such as lagomorph, avian, bovine, porcine, human, etc. may be obtained.Mammalian, especially primate, mGluR7 are of particular interest. Ifpartial clones are obtained, it is necessary to join them in properreading frame to produce a full length clone, using such techniques asendonuclease cleavage, ligation and loopout mutagenesis. It will beunderstood that the present invention includes degenerate polynucleotidesequences encoding amino acid sequences as described above.

A DNA sequence encoding mGluR7 is inserted into a suitable expressionvector, which in turn is used to transfect eukaryotic cells forexpression of mGluR7 or specific fragments thereof. Expression vectorsfor use in carrying out the present invention will comprise a promotercapable of directing the transcription of a cloned DNA and atranscriptional terminator. Depending on the host cell chosen,expression vectors may contain additional elements, such as one or moreorigins of replication, one or more selectable markers, enhancers, etc.Expression vector design is within the level of ordinary skill in theart. Expression vectors are also available from commercial suppliers.

To direct mGluR7 proteins of the present invention for transport to theplasma membrane, at least one signal sequence is operably linked to theDNA sequence of interest. The signal sequence may be derived from themGluR7 coding sequence, from other signal sequences described in theart, or synthesized de novo.

Host cells for use in practicing the present invention includemammalian, avian, plant, insect and fungal cells, but preferablymammalian cells. Fungal cells, including species of yeast (e.g.,Saccharomyces spp., particularly S. cerevisiae, Schizosaccharomycesspp.) or filamentous fungi (e.g., Aspergillus spp., Neurospora spp.) maybe used as host cells within the present invention. Suitable yeastvectors for use in the present invention include YRp7 (Struhl et al.,Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1978), YEp13 (Broach et al.,Gene 8: 121-133, 1979), POT vectors (Kawasaki et al, U.S. Pat. No.4,931,373, which is incorporated by reference herein), pJDB249 andpJDB219 (Beggs, Nature 275:104-108, 1978) and derivatives thereof. Suchvectors will generally include a selectable marker, which may be one ofany number of genes that exhibit a dominant phenotype for which aphenotypic assay exists to enable transformants to be selected.Preferred selectable markers are those that complement host cellauxotrophy, provide antibiotic resistance or enable a cell to utilizespecific carbon sources. Additional vectors, promoters and terminatorsfor use in expressing the receptor of the invention in yeast are wellknown in the art and are reviewed by, for example, Emr, Meth. Enzymol.185:231-279, (1990), incorporated herein by reference.

A variety of higher eukaryotic cells may serve as host cells forexpression of the mGluR7, although not all cell lines will be capable offunctional coupling of the receptor to the cell's second messengersystems. Cultured mammalian cells, such as BHK, CHO, Y1 (Shapiro et al.,TIPS Suppl. 43-46 (1989)), NG108-15 (Dawson et al., NeuroscienceApproached Through Cell Culture, Vol. 2, pages 89-114 (1989)), N1E-115(Liles et al., J. Biol. Chem. 261:5307-5313 (1986)), PC 12 and COS-1(ATCC CRL 1650) are preferred. Preferred BHK cell lines; are the tk⁻ts13 BHK cell line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA79:1106-1110 (1982)) and the BHK 570 cell line (deposited with theAmerican Type Culture Collection, 12301 Parklawn Dr., Rockville, Md.,under accession number CRL 10314). A tk⁻ BHK cell line is available fromthe ATCC under accession number CRL 1632.

Mammalian expression vectors for use in carrying out the presentinvention will include a promoter capable of directing the transcriptionof a cloned gene or cDNA. Preferred promoters include viral promotersand cellular promoters. Viral promoters include the immediate earlycytomegalovirus promoter (Boshart et al., Cell 41: 521-530, 1985) andthe SV40 promoter (Subramani et al., Mol. Cell. Biol. 1: 854-864, 1981).Cellular promoters include the mouse metallothionein-1 promoter(Palmiter et al., U.S. Pat. No. 4,579,821), a mouse V_(K) promoter(Bergman et al., Proc. Natl. Acad. Sci. USA 81: 7041-7045, 1983; Grantet al., Nuc. Acids Res. 15: 5496, 1987), a mouse V_(H) promoter (Loh etal., Cell 33: 85-93, 1983) and the major late promoter from adenovirus 2(Kaufman and Sharp, Mol. Cell. Biol. 2: 1304-13199, 1982). Suchexpression vectors may also contain a set of RNA splice sites locateddownstream from the promoter and upstream from the DNA sequence encodingthe peptide or protein of interest. Preferred RNA splice sites may beobtained from adenovirus and/or immunoglobulin genes. Also contained inthe expression vectors is a polyadenylation signal located downstream ofthe coding sequence of interest. Polyadenylation signals include theearly or late polyadenylation signals from SV40 (Kaufman and Sharp,ibid.), the polyadenylation signal from the adenovirus 5 E1B region andthe human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res.9: 3719-3730, 1981). The expression vectors may include a noncodingviral leader sequence, such as the adenovirus 2 tripartite leader,located between the promoter and the RNA splice sites. Vectors may alsoinclude enhancer sequences, such as the SV40 enhancer and the mouse μenhancer (Gillies, Cell 33: 717-728, 1983). Expression vectors may alsoinclude sequences encoding the adenovirus VA RNAs.

Cloned DNA sequences may be introduced into cultured mammalian cells by,for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603,1981; Graham and Van der Eb, Virology 52: 456, 1973.) Other techniquesfor introducing cloned DNA sequences into mammalian cells, such aselectroporation (Neumann et al., EMBO J. 1: 841-845, 1982), may also beused. In order to identify cells that have integrated the cloned DNA, aselectable marker is generally introduced into the cells along with thegene or cDNA of interest. Preferred selectable markers for use incultured mammalian cells include genes that confer resistance to drugs,such as neomycin, hygromycin, and methotrexate. The selectable markermay be an amplifiable selectable marker. Preferred amplifiableselectable markers are the DHFR gene and the neomycin resistance gene.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the mGluR7 gene of interest, or they may beintroduced on the same plasmid. If on the same plasmid, the selectablemarker and the mGluR7 gene can be under the control of differentpromoters or the same promoter, the latter arrangement producing adicistronic message. Constructs of this type are known in the art (forexample, Levinson and Simonsen, U.S. Pat. No. 4,713,339). It may also beadvantageous to add additional DNA, known as "carrier DNA" to themixture which is introduced into the cells.

Transfected mammalian cells are allowed to grow for a period of time,typically 1-2 days, to begin expressing the mGluR7 DNA sequence(s) ofinterest. Drug selection is then applied to select for growth of cellsthat are expressing the selectable marker in a stable fashion. For cellsthat have been transfected with an amplifiable selectable marker thedrug concentration may be increased in a stepwise manner to select forincreased copy number of the cloned sequences, thereby increasingexpression levels.

Promoters, terminators and methods suitable for introducing expressionvectors encoding recombinant mGluR7 into plant, avian and insect cellsare widely available. The use of baculoviruses, for example, as vectorsfor expressing heterologous DNA sequences in insect cells has beenreviewed by Atkinson et al. (Pestic. Sci. 28: 215-224, 1990). The use ofAgrobacterium rhizogenes as vectors for expressing genes in plant cellshas been reviewed by Sinkar et al. (J. Biosci. (Banglaore) 11: 47-58,1987).

Host cells containing DNA constructs of the present invention are thencultured to produce recombinant mGluR7. The cells are cultured accordingto accepted methods in a culture medium containing nutrients requiredfor growth of mammalian or other host cells. The growth medium willgenerally select for cells containing the DNA construct by, for example,drug selection or deficiency in an essential nutrient which iscomplemented by the selectable marker on the DNA construct orco-transfected with the DNA construct.

The mGluR7 produced according to the present invention may be purifiedfrom the recombinant expression systems or other sources usingpurification protocols that employ techniques generally available tothose skilled in the art. The most convenient sources for obtaininglarge quantities of mGluR7 are cells which express the recombinantreceptor protein. However, other sources, such as tissues, particularlytissues of the thalamus, neocortex and hypothalamus which contain thehighest levels of mGluR7, may also be employed.

Purification of mGluR7 can be achieved by conventional chemicalpurification means, such as liquid chromatography, lectin affinitychromatography, gradient centrifugation, and gel electrophoresis, amongothers. Methods of protein purification are known in the art (seegenerally, Scopes, R., Protein Purification, Springer-Verlag, NY (1982),which is incorporated herein by reference) and may be applied to thepurification of the mGluR7 and particularly the recombinantly producedmGluR7 described herein. In a preferred embodiment immunoaffinitychromatography is employed using antibodies directed against mGluR7 asherein described. In another method of purification, the isolatedpolynucleotide encoding mGluR7 or portions thereof can be modified atthe coding region for the amino terminus, just behind a signal sequence,with a sequence coding for a small hydrophilic peptide, such asdescribed in U.S. Pat. Nos. 4,703,004 and 4,782,137, which areincorporated herein by reference. Specific antibodies for the peptidefacilitate rapid purification of mGluR7, and the short peptide can thenbe removed with enterokinase.

Thus, as discussed above, the present invention provides mGluR7 isolatedfrom its natural cellular environment, substantially free of other Gprotein coupled glutamate receptors. However, mGluR7 of the inventionproduced by recombinant techniques can be coexpressed with other mGluRreceptors, or added as a component of an admixture of other receptors.Purified mGluR7 is also provided. Substantially pure mGluR7 of at leastabout 50% is preferred, at least about 70-80% more preferred, and 95-99%or more homogeneity most preferred, particularly for pharmaceuticaluses. Once purified, partially or to homogeneity, as desired, therecombinant mGluR7 or native mGluR7 may then be used to generateantibodies, in assay procedures, etc.

In another aspect, the invention concerns polypeptides and fragments ofmGluR7. Polypeptides and fragments of mGluR7 may be isolated fromrecombinant expression systems or may be synthesized by the solid phasemethod of Merrifield, Fed. Proc. 21:412 (1962), Merrifield, J. Am. Chem.Soc. 85:2149 (1963), or Barany and Merrifield, in The Peptides, vol. 2,pp. 1-284 (1979) Academic Press, NY, each of which are incorporatedherein by reference, or by use of an automated peptide synthesizer. By"polypeptides" is meant a sequence of at least about 3 amino acids,typically 6 or more, up to 100-200 amino acids or more, including entireproteins. For example, the portion(s) of mGluR7 protein which bindsligand may be identified by a variety of methods, such as by treatingpurified receptor with a protease or a chemical agent to fragment it anddetermine which fragment is able to bind to labeled glutamate in aligand blot. Alternatively, ligand-binding regions of mGluR7 proteinsmay be determined by sequence alignment of mGluR7 amino-terminal domainswith those of bacterial leucine-, isoleucine-, and valine-bindingproteins as described in O'Hara et al., Neuron 11:41-52 (1993),incorporated herein by reference. Polypeptides may then be synthesizedand used as antigen, to inhibit ligand-mGluR7 interaction, etc. Itshould be understood that as used herein, reference to mGluR7 is meantto include the protein, polypeptides, and fragments thereof unless thecontext indicates otherwise.

In another aspect, the invention provides means for regulating themGluR7-ligand interaction, and thus treating, therapeutically and/orprophylactically, a disorder which can be linked directly or indirectlyto mGluR7 or to its ligands, such as glutamate and other endogenousexcitatory amino acids. By mGluR7 ligand is meant a molecule capable ofbeing bound by a ligand-binding domain of mGluR7, a mGluR7 analog, orchimeric mGluR7 which can be produced in accordance with procedures forproducing chimeric receptors as generally described in U.S. Pat. No.4,859,609, incorporated by reference herein. The ligand may bechemically synthesized or may occur in nature. Ligands may be groupedinto agonists and antagonists. Agonists are those molecules whosebinding to a receptor induces the response pathway within a cell.Antagonists are those molecules whose binding to a receptor blocks theresponse pathway within a cell. By virtue of having the receptor of theinvention, agonists or antagonists may be identified which stimulate orinhibit the interaction of ligand with mGluR7. With either agonists orantagonists the metabolism and reactivity of cells which express thereceptor are controlled, thereby providing a means to abate or in someinstances prevent the disorder.

Thus, the invention provides screening procedures for identifyingagonists or antagonists of events mediated by the ligand-mGluR7interaction. Such screening assays may employ a wide variety of formats,depending to some extent on which aspect of the ligand/receptor/Gprotein interaction is targeted. For example, such assays may bedesigned to identify compounds which bind to the receptor and therebyblock or inhibit interaction of the receptor with the ligand. Otherassays can be designed to identify compounds which can substitute forligand and therefore stimulate mGluR7-mediated intracellular pathways.Yet other assays can be used to identify compounds which inhibit orfacilitate the association of mGluR7 to G protein and thereby mediatethe cellular response to mGluR7 ligand.

In one functional screening assay, mammalian cell lines are used whichexpress mGluR7 that is functionally coupled to a mammalian G protein. Inthis assay, compounds are screened for their relative affinity asreceptor agonists or antagonists by comparing the relative receptoroccupancy to the extent of ligand induced stimulation or inhibition ofsecond messenger metabolism. Although stimulation of mGluR7 receptors(e.g., by L-AP4) inhibits cAMP production in transfected cells, thetransduction mechanism is not well defined. Inhibition of cAMP could bethe mechanism for L-AP4 mediated decreases in transmitter release, butit is also likely that alternative coupling mechanisms are involved. Thedecreases observed in cAMP in mGluR7-expressing BHK cells is consistentwith the involvement of a Gi/Go protein. The possible mechanism ofcoupling is offered only by way of explanation, not limitation.

The screening procedure can be used to identify reagents such asantibodies which specifically bind to the receptor and not to otherknown members of the GluR family, i.e., mGluR1-mGluR6, and substantiallyaffect its interaction with ligand, for example. The antibodies may bemonoclonal or polyclonal, in the form of antiserum or monospecificantibodies, such as purified antiserum or monoclonal antibodies (e.g.,murine, humanized, or completely human) or mixtures thereof. Theantibodies which bind mGluR7 may be produced by a variety of well knownmethods.

In other embodiments, the invention provides screening assays conductedin vitro with cells which express the receptor. For example, the DNAwhich encodes the receptor or selected portions thereof may betransfected into an established cell line, e.g., a mammalian cell linesuch as BHK or CHO, using procedures known in the art (see, e.g.,Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, which isincorporated herein by reference). The receptor is then expressed by thecultured cells, and selected agents are screened for the desired effecton the cell, separately or in conjunction with an appropriate ligandsuch as glutamate or L-AP4, for example. Means for amplifying nucleicacid sequences which may be employed to amplify the receptor or portionsthereof are described in U.S. Pat. Nos. 4,683,195 and 4,683,202,incorporated herein by reference.

In yet another aspect, the screening assays provided by the inventionrelate to transgenic mammals whose germ cells and somatic cells containa nucleotide sequence encoding mGluR7 protein or a selected portion ofthe receptor which, e.g., binds ligand, GTP binding protein, or thelike. There are several means by which a sequence encoding, for example,the human mGluR7 may be introduced into a non-human mammalian embryo,some of which are described in, e.g., U.S. Pat. No. 4,736,866, Jaenisch,Science 240-1468-1474 (1988) and Westphal et al., Annu. Rev. Cell Biol.5:181-196 (1989), which are incorporated herein by reference. Theanimal's cells then express the receptor and thus may be used as aconvenient model for testing or screening selected agonists orantagonists.

In another aspect the invention concerns diagnostic methods andcompositions. By having the mGluR7 molecule and antibodies thereto, avariety of diagnostic assays are provided. For example, antibodies(including monoclonal antibodies) to mGluR7 can be used to determine thepresence and/or concentration of receptor in selected cells or tissuesin an individual or culture of interest. These assays can be used in thediagnosis and/or treatment of diseases such as, for example, cerebralischemia, Parkinsons, senile dementia and other cognitive disorders,Huntington's chorea, amyotrophic lateral sclerosis, migraine, andothers.

Numerous types of immunoassays and histological techniques are availableand can be used to identify mGluR7 on the surface of cells. Seegenerally, Harlow and Lane, Antibodies, A Laboratory Manual, Cold SpringHarbor Publications, N.Y. (1988), and Cuello, Immunohistochemistry, JohnWiley & Sons, New York (1984), each incorporated by reference herein. Inone assay format mGluR7 is identified and/or quantified by using labeledantibodies, preferably monoclonal antibodies which are reacted witholfactory bulb or brain tissues, e.g., cortex, striatum, hippocampus,cerebellum, and determining the specific binding thereto, the assaytypically being performed under conditions conducive to immune complexformation. Unlabeled primary antibody can be used in combination withlabels that are reactive with primary antibody to detect the receptor.For example, the primary antibody may be detected indirectly by alabeled secondary antibody made to specifically detect the primaryantibody. Alternatively, the anti-mGluR7 antibody can be directlylabeled. A wide variety of labels may be employed, such asradionuclides, particles (e.g., gold, ferritin, magnetic particles, redblood cells), fluorophores, chemiluminescers, enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors, ligands (particularlyhaptens), etc.

The mGluR7 DNA may be directly detected in cells with a labeled mGluR7DNA or synthetic oligonucleotide probe in a hybridization proceduresimilar to the Southern or dot blot. Also, the polymerase chain reaction(Saiki et al., Science 239:487 (1988), and U.S. Pat. No. 4,683,195) maybe used to amplify DNA sequences, which are subsequently detected bytheir characteristic size on agarose gels, Southern blot of these gelsusing mGluR7 DNA or a oligonucleotide probe, or a dot blot using similarprobes. The probes may comprise from about 14 nucleotides to about 25 ormore nucleotides, preferably, 40 to 60 nucleotides, and in someinstances a substantial portion or even the entire cDNA of mGluR7 may beused. The probes are labeled to provide a detectable signal, such aswith an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer,paramagnetic particle, etc.

Kits can also be supplied for use with the receptor of the subjectinvention in the detection of the presence of the receptor or antibodiesthereto, as might be desired in the case of autoimmune disease. Thus,antibodies to mGluR7, preferably monospecific antibodies such asmonoclonal antibodies, or compositions of the receptor may be provided,usually in lyophilized form in a container, either segregated or inconjunction with additional reagents, such as anti-antibodies, labels,gene probes, polymerase chain reaction primers and polymerase, and thelike.

The following examples are offered by way of illustration of theinvention and not by limitation.

EXAMPLE I cDNA Cloning and Sequencing of mGluR7

This Example describes screening a rat olfactory bulb cDNA library andidentification of a metabotropic receptor designated "mGluR7".

Degenerate oligonucleotide primers were used to amplify a region offirst strand olfactory bulb cDNA between transmembrane region II andintracellular loop III of mGluR cDNAs. The sequence of the 5' primer wasGC(TCAG)GG(TCAG)AT(ACT)TT(CT)(CT)T(TCAG)(GT)G(TCAG) (SEQ ID NO:4) andthe 3' primer was AT(TCAG)(GT)(AG)(CT)TT(TCAG)GC(CT)TC(AG)TT(AG)AA (SEQID NO:5). The 5' ends of both primers also contained sequences for anEcoRI restriction site. The PCR samples were analyzed by agarose gel andamplified fragments of approximately 350 basepairs were digested withEcoRI and cloned into pBluescript II KS (+) Stratagene Cloning Systems,La Jolla, Calif. Of sixty-five clones initially identified, three PCRproducts (Olf 1, 2 and 8) were identified as unique receptors, shown byDNA sequence analysis (Sanger et al., Proc. Natl. Acad. Sci. USA 74:5436-5367 1977!) to be distinct from previously isolated mGluR cDNAs.

The Olf 1, 2 and 8 clones were used as template to generate ³² P-labeledin vitro RNA transcripts. These were equally mixed and used to probe anadult rat olfactory bulb cDNA library (Stratagene, La Jolla, Calif.).Twenty-four duplicate positive clones were rescued by coinfection withhelper bacteriophage into pBluescript SK (-) Stratagene Cloning Systems,La Jolla, Calif.; fourteen had cDNA inserts as shown by restrictionanalysis. Southern blot analysis (Southern, J. Mol. Biol. 98: 503-5171975!) with Olf 1, 2 or 8 identified three distinct groups of clones:Olf 1 labeled 10 clones identified as mGluR5 (Abe et al., J. Biol. Chem.267: 13361-13368 1992!). Olf 8 identified 3 clones that also showedunique DNA sequence related to the mGluR family. Olf 2 labeled a singleclone that appeared to be a new member of the mGluR family. Thefull-length clone isolated with Olf 2 was designated "mGluR7."

The nucleotide sequence and predicted amino acid sequence for mGluR7 isshown in FIG. 1 (SEQ ID NO:1). The cDNA sequence contained an openreading frame of 2745 bp and an initiation codon consensus sequencesurrounding the presumed initiator methionine (Kozak, Nucl. Acids Res.15: 8125-8148 1987!). The deduced amino acid sequence of mGluR7 is 915amino acids with an estimated molecular weight of 125,126 kD.Hydrophobicity analysis (Kyte and Doolittle, J. Mol. Biol. 157: 105-1321982!) indicates a seven transmembrane-domain receptor with thecharacteristic features of the metabotropic receptors, including a largeamino-terminus domain of 590 amino acids and a large second cytoplasmicloop that appears to be involved in coupling of mGluRs to G proteins(Pin, Func. Neurol. supp. 8: 42 1993!). There are four putativeN-glycosylation sites in the amino terminus and one putativecalcium/calmodulin-dependent protein kinase II phosphorylation site inthe second intracellular domain.

Amino acid sequence comparison of mGluR7 and other members of the mGluRfamily showed a high degree of conservation with mGluR4 (69%) and mGluR6(67%), with a lower degree of conservation with mGluR1 (42%), mGluR2(45%), mGluR3 (45%) or mGluR5 (45%). These overall homologies placemGluR7 into a subset of mGluRs that includes mGluR4 and mGluR6.

EXAMPLE II Expression of mGluR7

BHK cells were transfected with mGluR7 subcloned into pZEM229R. PlasmidpZEM229R is a pUC18-based expression vector containing an EcoRI cloningsite between a mouse metallothionein-I promoter and an SV40transcription terminator. The vector also contains an expression unit ofthe SV40 early promoter, mouse dihydrofolate reductase gene, and SV40terminator. The two expression units are in opposite orientations withtheir respective promoters adjacent to each other. E coli HB101transformed with pZEM229R was deposited with American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. on Sep. 28, 1993.

The cells were grown in 10% fetal calf serum and Dulbecco's minimalessential media (DMEM), and transfected clones were selected bymethotrexate resistance. Resistant clones were assayed for the presenceof mGluR7 RNA and protein. Confluent cells were assayed for cAMPproduction (Amersham) in the presence of IBMX (1 mM)isobutylmethylxanthine; cAMP levels were measured after 10 minutetreatment with forskolin (10 μM) in the presence or absence of mGluRagonists or antagonists. Assays were performed in triplicate three timesand the average value from these experiments expressed as the mean cAMPlevels plus and minus the standard deviation. The basal cAMP productionwas subtracted from the stimulated levels and the inhibition was plottedas a percent of the forskolin-stimulated control.

For oocyte expression studies, Xenopus laevis (Xenopus I, Ann Arbor)were anesthetized with tricaine and oocytes harvested using steriletechnique. RNA transcripts from mGluR1 and mGluR7 were injected intoStage V-VI oocytes (1-100 ng RNA/oocyte) and the oocytes were assayedfor production of a calcium-activated chloride current using atwo-electrode voltage clamp. Harvesting of oocytes, solutions andrecording methods were as described by Lester and colleagues (Dascal etal., Brain Res. 387: 201-209 1986!, which is incorporated herein byreference).

In several experiments, oocytes injected with mGluR7 mRNA evoked noglutamate responses, despite large calcium-activated chloride currentsin sister oocytes injected with mGluR1 mRNA. These data suggested thatmGluR7, unlike mGluR1 and mGluR5 (Masu et al., Nature 349: 760-7651991!; Houamed et al., Science 252: 1318-1321 1991!; and Abe et al.,supra), does not activate phospholipase C.

Other metabotropic receptors (mGluR2-4 and mGluR6) have been shown toinhibit cAMP production in Chinese hamster ovary (CHO) cells (Tanabe etal., Neuron 8: 169-179 1992!; Tanabe et al., J. Neurosci. 13: 1372-13781993!; and Nakajima et al., J. Biol. Chem. 268: 11868-11873 1993!), orBHK cells (Thomsen et al., Eur. J. Pharmacol. 227: 361-362 1992!). Asshown in FIG. 2, L-AP4 (1 mM) or glutamate (1 mM) inhibitedforskolin-stimulated cAMP production in BHK cells stably expressingmGluR7 cDNA, confirming that mGluR7 is a member of the metabotropicreceptor family. The presence of mGluR7 in the transfected BHK cells wasconfirmed by RNA blot, and by immunoblot analysis using a polyclonalantisera prepared against an mGluR7 peptide representing amino acids896-909 of mGluR7(Val-Asp-Pro-Asn-Ser-Pro-Ala-Ala-Lys-Lys-Lys-Tyr-Val-Ser--SEQ ID NO:3).

L-AP4 had no significant effect on basal cAMP levels or onforskolin-stimulated cAMP levels in wild-type BHK cells. Lowerconcentrations of L-AP4 (100 μM) or glutamate (100 μM) produced smalldecreases in cAMP which were not statistically significant. ACPD (1 mM)or quisqualate (0.5 mM) were much less effective at concentrations farin excess of those that result in maximal stimulation of mGluR1.Likewise, 2-amino-3-phosphonopropionic acid (L-AP3; 3 mM) orRS-α-methyl-4-carboxyphenylglycine (MCPG; 1 mM) that antagonize mGluR1in some preparations did not antagonize mGluR7-mediated cAMP inhibition.L-AP4 (1 mM) stimulation of a BHK clone expressing mGluR4 produced 45%inhibition, confirming the sensitivity of the assay. Although thesubmaximal inhibition of CAMP by mGluR7 obtained prevented fullconcentration-response analysis, L-AP4 and glutamate were much lesspotent agonists of mGluR7 in these experiments than has previously beenobserved for mGluR4 or mGluR6. The modest inhibition at lower levels ofagonist observed in these experiments may be due either to lower levelsof receptor expression or to less efficient coupling to adenylatecyclase for mGluR7. cAMP inhibition may not be the principaltransduction mechanism for mGluR7 in neurons, as mGluR6 inhibits cAMP inCHO cells, but its primary action in neurons is likely to be stimulationof a phosphodiesterase (Nawy and Jahr, Nature 346: 269-271 1990!).

EXAMPLE III Distribution of mGluR7 RNA

A mGluR7 cDNA probe was hybridized to poly (A)+RNA isolated from severalregions of adult rat brain to determine distribution of mGluR7 RNA.Total RNA was isolated from freshly dissected rat brain regionsrepresenting cerebellum, hippocampus, hypothalamus, olfactory bulb,brainstem, midbrain, thalamus, and cortex as described (Chomczynski andSacchi, Anal. Biochem. 162: 156-159 1987!). Poly (A)+RNA was isolatedusing a magnesphere RNA isolation kit (Promega). Two micrograms ofpoly(A)+RNA sample were fractionated by denaturing agarose gelelectrophoresis and capillary transferred to nitrocellulose, followed byhybridization with a random-primed ³² P-labeled 719 bp XhoI fragmentcontaining nucleotides 1-197 of the mGluR7 cDNA and a 1234 bp XhoI/PstIfragment of mGluR4 containing nucleotides 791-2025.

Hybridization of a mGluR7 cDNA probe to poly (A)+RNA isolated fromseveral regions of adult rat brain revealed a single class of messengerRNA of approximately 4.4 Kb. The previously identified L-AP4-sensitivereceptors mGluR4 and mGluR6 show quite constricted patterns of RNAexpression. However, mGluR7 was widely expressed in the CNS. Highestexpression was seen in the thalamus, neocortex and hypothalamus, withsignificant levels of expression in the hippocampus, olfactory bulb,brainstem and midbrain.

For in situ hybridization, adult Sprague-Dawley rats (200-250 g) wereanesthetized with pentobarbital and perfused transcardially withice-cold saline, followed by ice-cold fixative (4% paraformaldehyde/0.1Msodium borate, pH 9.5). The brains were post-fixed overnight in fixativecontaining 10% sucrose. Sections (25 μm) were mounted on gelatin- andpoly-L-lysine- coated glass, fixed for 15 minutes in 4%paraformaldehyde/0.1M phosphate buffered saline (PBS), washed twice in0.1M PBS, and treated for 30 min at 37° C. in proteinase K (0.001% in0.1M Tris/0.05M EDTA, pH 8) followed by 0.0025% acetic anhydride in 0.1Mtriethanolamine at room temperature followed by dehydration. A 719 bpXhoI fragment containing nucleotides 1-197 of mGluR7 and a 1234 bpXhoI/PstI fragment of mGluR4 containing nucleotides 791-2025 weresubcloned into pBluescript KS(+). ³⁵ S-labeled antisense RNA wastranscribed from each template and used for hybridization at 10⁷ cpm/mlfor 20 hours at 58° C. in hybridization buffer (Mountjoy et al., Science257: 1248-1251 1992!). Slides were processed as described (Simerly andYoung, Mol. Endocrinol. 5: 424-432 1991!) then dipped in NTB-2 liquidphotographic emulsion (Kodak), exposed for 13 days, developed with D-19developer and counterstained with thionin.

In situ hybridization with a mGluR7 probe was consistent with the RNAblot analysis showing a broad distribution of hybridization in cortex,olfactory bulb, hippocampus, thalamus and caudate putamen, but withrelatively low signal in the cerebellum. The distribution of mGluR7 wasmore widespread than mGluR4.

Comparison of mGluR7 with mGluR4 revealed complementary patterns ofhybridization in the hippocampus and olfactory bulb, although both werehighly expressed in the entorhinal cortex, subiculum and presubiculum.There was strong hybridization of mGluR7 in the dentate gyrus andhippocampus (CA1-CA4) and in the mitral/tufted cells of the olfactorybulb. By contrast, mGluR4 was present in the glomerular and granularcell layers of the olfactory bulb. In the cerebellum, mGluR4 mRNA waspresent in the granular layers while mGluR7 was restricted to thePurkinje cells.

The existence of multiple metabotropic receptors with distinct, butoverlapping patterns of distribution suggested that single cells maycontain more than one mGluR subtype. This was tested in the olfactorybulb by a double labeling method using probes for mGluR7 and mGluR1. Fordouble labeling, mGluR1 probes were labeled with digoxigenin-UTP asabove whereas mGluR7 probes were labeled with ³⁵ S-UTP.Digoxigenin-labeled in vitro RNA transcripts for mGluR1 were generatedfrom a 1363 bp EcoRI/SacI cDNA fragment containing nucleotides 361-1724of mGluR1 (Masu et al., Nature 349: 760-765 (1991)). Sections wereincubated with both probes, reacted for digoxigenin and processed asabove. After incubation overnight in 2X SSC/0.05% Triton X-100/2% normalgoat serum, sections were incubated with anti-digoxigenin alkalinephosphatase conjugate diluted 1:1000 in 0.1M Tris-HCl pH 7.5/0.15MNaCl/0.3% Triton X-100/1% normal goat serum for 5 hrs, followed byincubation overnight in the dark in 0.1M Tris-HCl pH 9.5/0.1M NaCl/0.05MMgCl₂ containing 337.5 mg/ml 4-nitro blue tetrazolium chloride/175 mg/ml5-bromo-4-chloro-3-indolyl-phosphate/300 mg/ml levamisole. The reactionwas stopped with 0.01M Tris-HCl pH 8.1/1 mM EDTA, followed by rapiddehydration in increasing concentrations of ethanol containing 0.1XSSC/1 mM dithiothreitol. After vacuum drying, slides were dipped inIlford K.5 photographic emulsion and exposed for 13 days, followed bydevelopment with D-19. Slides were not counter-stained.

Most mitral/tufted cells were labeled with mGluR7 as represented by thesilver grains. Likewise all the mGluR7 labeled cells were also labeledwith mGluR1 using digoxigenin as the chromogen. The sections were notcounterstained and thus the diffuse staining of the soma represents thedigoxigenin reaction product. The mGluR1 has been localized to bothpostsynaptic and presynaptic locations (Martin et al., Neuron 9: 259-270(1992) and unpublished data) whereas the effects of L-AP4 onmitral/tufted cells is presynaptic (Trombley and Westbrook, J. Neurosci.12: 2-43-2050 (1992)). This may suggest a compartmentalization ofdifferent mGluR subtypes within the same neuron. Thus, theL-AP4-sensitivity and expression pattern of mGluR7 suggest that it is apresynaptic autoreceptor in selected CNS pathways.

All publications and patents mentioned in this specification are hereinincorporated by reference into the specification to the same extent asif each individual publication or patent was specifically andindividually indicated to be incorporated herein by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2997 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 133..2877                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ACCCTCTGTGCCTACAGAAGTCCCTGGGCTTCCCCCAAAGGAGCTCACCTCCAGAAGCCC60                GGACCTGGGAGAGCCCACCAGCATACCCTCCAGCGCCGCCGCCGCCGCTACCGCAGCAGC120               ATCCGGAGCGGCATGGTCCAGCTGGGGAAGCTGCTCCGCGTCCTGACT168                           MetValGlnLeuGlyLysLeuLeuArgValLeuThr                                          1510                                                                          TTGATGAAGTTCCCCTGCTGCGTGCTGGAGGTGCTCCTGTGCGTGCTG216                           LeuMetLysPheProCysCysValLeuGluValLeuLeuCysValLeu                              152025                                                                        GCGGCGGCGGCGCGCGGCCAGGAGATGTACGCCCCGCACTCGATCCGG264                           AlaAlaAlaAlaArgGlyGlnGluMetTyrAlaProHisSerIleArg                              303540                                                                        ATCGAGGGGGACGTCACCCTTGGGGGGTTGTTCCCAGTGCACGCCAAG312                           IleGluGlyAspValThrLeuGlyGlyLeuPheProValHisAlaLys                              45505560                                                                      GGTCCCAGCGGAGTGCCCTGCGGCGACATCAAGAGGGAGAATGGGATC360                           GlyProSerGlyValProCysGlyAspIleLysArgGluAsnGlyIle                              657075                                                                        CACAGGCTGGAAGCTATGCTTTATGCCCTGGACCAGATCAACAGCGAT408                           HisArgLeuGluAlaMetLeuTyrAlaLeuAspGlnIleAsnSerAsp                              808590                                                                        CCCAACCTGCTGCCCAATGTAACGTTAGGCGCGCGGATCCTGGACACT456                           ProAsnLeuLeuProAsnValThrLeuGlyAlaArgIleLeuAspThr                              95100105                                                                      TGTTCCAGGGACACTTATGCGCTCGAACAGTCGCTCACTTTCGTCCAG504                           CysSerArgAspThrTyrAlaLeuGluGlnSerLeuThrPheValGln                              110115120                                                                     GCGCTTATCCAGAAGGACACCTCCGACGTGCGTTGCACCAACGGAGAG552                           AlaLeuIleGlnLysAspThrSerAspValArgCysThrAsnGlyGlu                              125130135140                                                                  CCCCCGGTTTTCGTCAAGCCAGAGAAAGTAGTTGGAGTGATTGGGGCT600                           ProProValPheValLysProGluLysValValGlyValIleGlyAla                              145150155                                                                     TCGGGGAGCTCCGTCTCCATCATGGTAGCCAACATCTTGAGGCTTTTT648                           SerGlySerSerValSerIleMetValAlaAsnIleLeuArgLeuPhe                              160165170                                                                     CAGATCCCTCAGATTAGTTATGCATCCACGGCTCCTGAACTCAGTGAT696                           GlnIleProGlnIleSerTyrAlaSerThrAlaProGluLeuSerAsp                              175180185                                                                     GACCGGCGCTATGACTTCTTCTCTCGAGTGGTCCCGCCTGATTCCTTC744                           AspArgArgTyrAspPhePheSerArgValValProProAspSerPhe                              190195200                                                                     CAAGCCCAGGCGATGGTTGACATTGTAAAGGCTTTGGGCTGGAATTAC792                           GlnAlaGlnAlaMetValAspIleValLysAlaLeuGlyTrpAsnTyr                              205210215220                                                                  GTGTCTACTCTTGCATCTGAAGGAAGCTATGGAGAGAAAGGTGTGGAG840                           ValSerThrLeuAlaSerGluGlySerTyrGlyGluLysGlyValGlu                              225230235                                                                     TCCTTCACACAGATTTCCAAAGAGGCAGGTGGGCTCTGCATTGCCCAG888                           SerPheThrGlnIleSerLysGluAlaGlyGlyLeuCysIleAlaGln                              240245250                                                                     TCCGTGAGAATCCCCCAAGAGCGCAAAGACAGGACCATTGACTTTGAT936                           SerValArgIleProGlnGluArgLysAspArgThrIleAspPheAsp                              255260265                                                                     AGAATTATCAAACAGCTCTTGGACACTCCCAACTCCAGGGCCGTCGTG984                           ArgIleIleLysGlnLeuLeuAspThrProAsnSerArgAlaValVal                              270275280                                                                     ATTTTTGCCAACGATGAGGATATAAAGCAGATCCTTGCCGCCGCCAAA1032                          IlePheAlaAsnAspGluAspIleLysGlnIleLeuAlaAlaAlaLys                              285290295300                                                                  AGAGCTGACCAAGTAGGCCATTTTCTCTGGGTCGGGTCAGACAGCTGG1080                          ArgAlaAspGlnValGlyHisPheLeuTrpValGlySerAspSerTrp                              305310315                                                                     GGTTCCAAAATCAACCCACTGCATCAGCACGAAGATATTGCGGAAGGA1128                          GlySerLysIleAsnProLeuHisGlnHisGluAspIleAlaGluGly                              320325330                                                                     GCCATAACAATCCAGCCTAAAAGGGCAACCGTGGAAGGATTTGATGCT1176                          AlaIleThrIleGlnProLysArgAlaThrValGluGlyPheAspAla                              335340345                                                                     TACTTCACATCCCGGACACTTGAAAACAACAGGAGAAATGTATGGTTT1224                          TyrPheThrSerArgThrLeuGluAsnAsnArgArgAsnValTrpPhe                              350355360                                                                     GCCGAATACTGGGAAGAAAACTTCAACTGCAAGTTGACAATTAGTGGG1272                          AlaGluTyrTrpGluGluAsnPheAsnCysLysLeuThrIleSerGly                              365370375380                                                                  TCCAAAAAAGAAGACACAGATCGCAAATGCACAGGACAGGAGCGAATT1320                          SerLysLysGluAspThrAspArgLysCysThrGlyGlnGluArgIle                              385390395                                                                     GGAAAAGACTCCAATTATGAGCAGGAAGGTAAAGTACAGTTTGTGATT1368                          GlyLysAspSerAsnTyrGluGlnGluGlyLysValGlnPheValIle                              400405410                                                                     GATGCTGTCTATGCCATGGCCCATGCTCTTCATCACATGAACAAGGAT1416                          AspAlaValTyrAlaMetAlaHisAlaLeuHisHisMetAsnLysAsp                              415420425                                                                     CTGTGTGCTGACTACCGCGGAGTGTGCCCAGAGATGGAGCAAGCAGGC1464                          LeuCysAlaAspTyrArgGlyValCysProGluMetGluGlnAlaGly                              430435440                                                                     GGCAAGAAGTTGTTGAAGTATATCCGCCATGTTAACTTCAATGGTAGT1512                          GlyLysLysLeuLeuLysTyrIleArgHisValAsnPheAsnGlySer                              445450455460                                                                  GCTGGAACCCCAGTAATGTTTAACAAAAATGGCGATGCTCCAGGGCGT1560                          AlaGlyThrProValMetPheAsnLysAsnGlyAspAlaProGlyArg                              465470475                                                                     TATGACATCTTCCAATACCAGACAACAAACACAACCAACCCTGGTTAT1608                          TyrAspIlePheGlnTyrGlnThrThrAsnThrThrAsnProGlyTyr                              480485490                                                                     CGTCTCATTGGGCAGTGGACAGATGAACTTCAGCTCAATATAGAGGAC1656                          ArgLeuIleGlyGlnTrpThrAspGluLeuGlnLeuAsnIleGluAsp                              495500505                                                                     ATGCAGTGGGGCAAAGGAGTCCGAGAGATCCCATCCTCTGTGTGTACA1704                          MetGlnTrpGlyLysGlyValArgGluIleProSerSerValCysThr                              510515520                                                                     TTGCCATGCAAGCCTGGGCAAAGGAAGAAGACACAGAAGGGAACGCCT1752                          LeuProCysLysProGlyGlnArgLysLysThrGlnLysGlyThrPro                              525530535540                                                                  TGCTGCTGGACCTGTGAGCCCTGTGATGGATACCAGTATCAGTTTGAT1800                          CysCysTrpThrCysGluProCysAspGlyTyrGlnTyrGlnPheAsp                              545550555                                                                     GAGATGACCTGTCAGCATTGTCCCTACGACCAGAGGCCCAATGAGAAC1848                          GluMetThrCysGlnHisCysProTyrAspGlnArgProAsnGluAsn                              560565570                                                                     CGAACTGGCTGTCAGAACATCCCAATCATCAAACTGGAGTGGCACTCC1896                          ArgThrGlyCysGlnAsnIleProIleIleLysLeuGluTrpHisSer                              575580585                                                                     CCCTGGGCTGTCATTCCTGTCTTCCTGGCAATGTTGGGGATCATTGCC1944                          ProTrpAlaValIleProValPheLeuAlaMetLeuGlyIleIleAla                              590595600                                                                     ACCATCTTTGTCATGGCAACTTTCATCCGCTACAATGACACACCCATT1992                          ThrIlePheValMetAlaThrPheIleArgTyrAsnAspThrProIle                              605610615620                                                                  GTCAGGGCATCTGGGCGGGAACTCAGCTATGTTTTATTGACAGGCATC2040                          ValArgAlaSerGlyArgGluLeuSerTyrValLeuLeuThrGlyIle                              625630635                                                                     TTTCTCTGCTATATCATCACCTTCCTAATGATTGCCAAACCAGATGTG2088                          PheLeuCysTyrIleIleThrPheLeuMetIleAlaLysProAspVal                              640645650                                                                     GCAGTGTGTTCTTTCCGACGTGTCTTCTTGGGCTTGGGCATGTGTATT2136                          AlaValCysSerPheArgArgValPheLeuGlyLeuGlyMetCysIle                              655660665                                                                     AGTTATGCTGCCCTTTTAACAAAGACCAATCGGATTTATCGCATATTC2184                          SerTyrAlaAlaLeuLeuThrLysThrAsnArgIleTyrArgIlePhe                              670675680                                                                     GAGCAGGGCAAGAAATCGGTGACAGCTCCCAGACTCATAAGCCCAACG2232                          GluGlnGlyLysLysSerValThrAlaProArgLeuIleSerProThr                              685690695700                                                                  TCACAACTGGCGATCACTTCCAGTTTAATATCGGTGCAGCTTCTAGGT2280                          SerGlnLeuAlaIleThrSerSerLeuIleSerValGlnLeuLeuGly                              705710715                                                                     GTCTTCATTTGGTTTGGGGTTGACCCCCCCAACATTATCATAGACTAC2328                          ValPheIleTrpPheGlyValAspProProAsnIleIleIleAspTyr                              720725730                                                                     GATGAGCATAAGACCATGAACCCAGAACAAGCAAGGGGTGTTCTCAAA2376                          AspGluHisLysThrMetAsnProGluGlnAlaArgGlyValLeuLys                              735740745                                                                     TGTGACATCACAGACCTTCAAATCATTTGTTCCCTGGGATATAGCATT2424                          CysAspIleThrAspLeuGlnIleIleCysSerLeuGlyTyrSerIle                              750755760                                                                     CTTCTCATGGTCACATGTACTGTGTATGCCATCAAGACTCGGGGCGTA2472                          LeuLeuMetValThrCysThrValTyrAlaIleLysThrArgGlyVal                              765770775780                                                                  CCAGAGAATTTTAATGAAGCCAAGCCCATTGGGTTCACTATGTACACG2520                          ProGluAsnPheAsnGluAlaLysProIleGlyPheThrMetTyrThr                              785790795                                                                     ACGTGTATCGTATGGCTTGCCTTCATCCCAATATTTTTTGGCACAGCG2568                          ThrCysIleValTrpLeuAlaPheIleProIlePhePheGlyThrAla                              800805810                                                                     CAGTCAGCAGAAAAGCTCTACATACAAACTACCACGCTTACAATCTCC2616                          GlnSerAlaGluLysLeuTyrIleGlnThrThrThrLeuThrIleSer                              815820825                                                                     ATGAACCTAAGTGCGTCAGTGGCGCTGGGAATGCTATACATGCCGAAA2664                          MetAsnLeuSerAlaSerValAlaLeuGlyMetLeuTyrMetProLys                              830835840                                                                     GTGTACATCATCATTTTCCACCCTGAACTCAATGTCCAGAAACGGAAG2712                          ValTyrIleIleIlePheHisProGluLeuAsnValGlnLysArgLys                              845850855860                                                                  CGAAGCTTCAAGGCCGTAGTCACAGCAGCCACCATGTCATCAAGGCTG2760                          ArgSerPheLysAlaValValThrAlaAlaThrMetSerSerArgLeu                              865870875                                                                     TCACACAAACCCAGTGACAGGCCCAACGGTGAGGCAAAGACAGAACTC2808                          SerHisLysProSerAspArgProAsnGlyGluAlaLysThrGluLeu                              880885890                                                                     TGTGAAAATGTAGACCCAAACAGCCCTGCTGCAAAAAAGAAGTATGTC2856                          CysGluAsnValAspProAsnSerProAlaAlaLysLysLysTyrVal                              895900905                                                                     AGTTATAATAACCTGGTTATCTAACCTGTTCCATGCCATGGAGCCACAGAG2907                       SerTyrAsnAsnLeuValIle                                                         910915                                                                        GAGGAAGACCTTCAGTTATTCTGTCACCCACGGTGGCATTGGACTCTTGGTCCTGCCCGC2967              TTCCTATCTCTGGAGGAGCTTCCTCGTGCC2997                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 915 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetValGlnLeuGlyLysLeuLeuArgValLeuThrLeuMetLysPhe                              151015                                                                        ProCysCysValLeuGluValLeuLeuCysValLeuAlaAlaAlaAla                              202530                                                                        ArgGlyGlnGluMetTyrAlaProHisSerIleArgIleGluGlyAsp                              354045                                                                        ValThrLeuGlyGlyLeuPheProValHisAlaLysGlyProSerGly                              505560                                                                        ValProCysGlyAspIleLysArgGluAsnGlyIleHisArgLeuGlu                              65707580                                                                      AlaMetLeuTyrAlaLeuAspGlnIleAsnSerAspProAsnLeuLeu                              859095                                                                        ProAsnValThrLeuGlyAlaArgIleLeuAspThrCysSerArgAsp                              100105110                                                                     ThrTyrAlaLeuGluGlnSerLeuThrPheValGlnAlaLeuIleGln                              115120125                                                                     LysAspThrSerAspValArgCysThrAsnGlyGluProProValPhe                              130135140                                                                     ValLysProGluLysValValGlyValIleGlyAlaSerGlySerSer                              145150155160                                                                  ValSerIleMetValAlaAsnIleLeuArgLeuPheGlnIleProGln                              165170175                                                                     IleSerTyrAlaSerThrAlaProGluLeuSerAspAspArgArgTyr                              180185190                                                                     AspPhePheSerArgValValProProAspSerPheGlnAlaGlnAla                              195200205                                                                     MetValAspIleValLysAlaLeuGlyTrpAsnTyrValSerThrLeu                              210215220                                                                     AlaSerGluGlySerTyrGlyGluLysGlyValGluSerPheThrGln                              225230235240                                                                  IleSerLysGluAlaGlyGlyLeuCysIleAlaGlnSerValArgIle                              245250255                                                                     ProGlnGluArgLysAspArgThrIleAspPheAspArgIleIleLys                              260265270                                                                     GlnLeuLeuAspThrProAsnSerArgAlaValValIlePheAlaAsn                              275280285                                                                     AspGluAspIleLysGlnIleLeuAlaAlaAlaLysArgAlaAspGln                              290295300                                                                     ValGlyHisPheLeuTrpValGlySerAspSerTrpGlySerLysIle                              305310315320                                                                  AsnProLeuHisGlnHisGluAspIleAlaGluGlyAlaIleThrIle                              325330335                                                                     GlnProLysArgAlaThrValGluGlyPheAspAlaTyrPheThrSer                              340345350                                                                     ArgThrLeuGluAsnAsnArgArgAsnValTrpPheAlaGluTyrTrp                              355360365                                                                     GluGluAsnPheAsnCysLysLeuThrIleSerGlySerLysLysGlu                              370375380                                                                     AspThrAspArgLysCysThrGlyGlnGluArgIleGlyLysAspSer                              385390395400                                                                  AsnTyrGluGlnGluGlyLysValGlnPheValIleAspAlaValTyr                              405410415                                                                     AlaMetAlaHisAlaLeuHisHisMetAsnLysAspLeuCysAlaAsp                              420425430                                                                     TyrArgGlyValCysProGluMetGluGlnAlaGlyGlyLysLysLeu                              435440445                                                                     LeuLysTyrIleArgHisValAsnPheAsnGlySerAlaGlyThrPro                              450455460                                                                     ValMetPheAsnLysAsnGlyAspAlaProGlyArgTyrAspIlePhe                              465470475480                                                                  GlnTyrGlnThrThrAsnThrThrAsnProGlyTyrArgLeuIleGly                              485490495                                                                     GlnTrpThrAspGluLeuGlnLeuAsnIleGluAspMetGlnTrpGly                              500505510                                                                     LysGlyValArgGluIleProSerSerValCysThrLeuProCysLys                              515520525                                                                     ProGlyGlnArgLysLysThrGlnLysGlyThrProCysCysTrpThr                              530535540                                                                     CysGluProCysAspGlyTyrGlnTyrGlnPheAspGluMetThrCys                              545550555560                                                                  GlnHisCysProTyrAspGlnArgProAsnGluAsnArgThrGlyCys                              565570575                                                                     GlnAsnIleProIleIleLysLeuGluTrpHisSerProTrpAlaVal                              580585590                                                                     IleProValPheLeuAlaMetLeuGlyIleIleAlaThrIlePheVal                              595600605                                                                     MetAlaThrPheIleArgTyrAsnAspThrProIleValArgAlaSer                              610615620                                                                     GlyArgGluLeuSerTyrValLeuLeuThrGlyIlePheLeuCysTyr                              625630635640                                                                  IleIleThrPheLeuMetIleAlaLysProAspValAlaValCysSer                              645650655                                                                     PheArgArgValPheLeuGlyLeuGlyMetCysIleSerTyrAlaAla                              660665670                                                                     LeuLeuThrLysThrAsnArgIleTyrArgIlePheGluGlnGlyLys                              675680685                                                                     LysSerValThrAlaProArgLeuIleSerProThrSerGlnLeuAla                              690695700                                                                     IleThrSerSerLeuIleSerValGlnLeuLeuGlyValPheIleTrp                              705710715720                                                                  PheGlyValAspProProAsnIleIleIleAspTyrAspGluHisLys                              725730735                                                                     ThrMetAsnProGluGlnAlaArgGlyValLeuLysCysAspIleThr                              740745750                                                                     AspLeuGlnIleIleCysSerLeuGlyTyrSerIleLeuLeuMetVal                              755760765                                                                     ThrCysThrValTyrAlaIleLysThrArgGlyValProGluAsnPhe                              770775780                                                                     AsnGluAlaLysProIleGlyPheThrMetTyrThrThrCysIleVal                              785790795800                                                                  TrpLeuAlaPheIleProIlePhePheGlyThrAlaGlnSerAlaGlu                              805810815                                                                     LysLeuTyrIleGlnThrThrThrLeuThrIleSerMetAsnLeuSer                              820825830                                                                     AlaSerValAlaLeuGlyMetLeuTyrMetProLysValTyrIleIle                              835840845                                                                     IlePheHisProGluLeuAsnValGlnLysArgLysArgSerPheLys                              850855860                                                                     AlaValValThrAlaAlaThrMetSerSerArgLeuSerHisLysPro                              865870875880                                                                  SerAspArgProAsnGlyGluAlaLysThrGluLeuCysGluAsnVal                              885890895                                                                     AspProAsnSerProAlaAlaLysLysLysTyrValSerTyrAsnAsn                              900905910                                                                     LeuValIle                                                                     915                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ValAspProAsnSerProAlaAlaLysLysLysTyrValSer                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCTCAGGGTCAGATACTTTCTCTTTCAGGTGTCAG35                                         (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ATTCAGGTAGCTTTTCAGGCCTTCAGTTAGAA32                                            __________________________________________________________________________

What is claimed is:
 1. An oligonucleotide probe which comprises anoligonucleotide molecule of at least 25 nucleotides in length whichprobe is capable of hybridizing under high stringency conditions with agene that encodes the metabotropic glutamate receptor mGluR7 accordingto SEQ ID NO:1 and not to one or more metabotropic glutamate receptorgenes mGluR₁, mGluR2, mGluR3, mGluR4, mGluR5 or mGluR6.
 2. The probe ofclaim 1, which comprises from about 40 to about 60 nucleotides inlength.
 3. The probe of claim 1, which is labeled to provide adetectable signal.
 4. The probe of claim 1, having a length of between60 nucleotides and up to the entire length of the mGluR7 cDNA of SEQ IDNO:1.
 5. The probe of claim 1, wherein the probe does not hybridizeunder high stringency conditions to mGluR4 or mGluR6.
 6. The probe ofclaim 1, wherein the probe is complementary to a portion of the cDNAsequence of SEQ ID NO:1 that encodes one or more amino acids associatedwith ligand binding.
 7. The probe of claim 6, wherein said one or moreamino acids associated with ligand binding are selected from the groupconsisting of amino acids 72, 157-163, 180-184, 197, 410, and 472 ofFIGS. 1-1 through 1-3.
 8. The probe of claim 1, wherein the probe iscomplementary to a portion of the cDNA sequence of SEQ ID NO:1 thatencodes one or more amino acids contained within a transmembrane domainof mGluR7.
 9. The probe of claim 8, wherein said one or more amino acidsare contained within amino acid segments I-VII of FIGS. 1-1 through 1-3.10. The probe of claim 1, wherein the probe is complementary to aportion of the cDNA sequence of SEQ ID NO:1 that encodes one or moreamino acids contained within an N-terminal (extracellular) domain ofmGluR7.
 11. The probe of claim 1, wherein the probe is complementary toa portion of the cDNA sequence of SEQ ID NO:1 that encodes one or moreamino acids contained within a C-terminal (cytoplasmic) domain ofmGluR7.